Neermala Ragoonanan

BIOL 2360 – Biochemistry IIA


What is metabolomics?

Metabolomics combines strategies to identify and quantify cellular metabolites using analytical methods such as gas chromatography, liquid chromatography and nuclear magnetic resonance spectroscopy. These analytical approaches are done to analyze different cell products such as those from gene expression (transcripts), proteins, and metabolites. All of these so-called ’omics approaches including genomics, transcriptomics, proteomics and metabolomics are considered important tools to be applied and utilized to understand the biology of an organism and its response to environmental stimuli or genetic perturbation.

There are four approaches in metabolomics: target analysis, metabolite profiling, metabolomics, and metabolic fingerprinting. Target analysis includes the determination and quantification of a small set of known metabolites (targets) using one particular analytical technique for the compounds of interest. Metabolite profiling aims at the analysis of a larger set of compounds, both identified and unknown with respect to their chemical nature. Metabolomics employs complementary analytical methodologies for example, LC-MS/MS, GC-MS, and/or NMR, in order to determine and quantify as many metabolites as possible, either identified or unknown compounds. Metabolic fingerprinting is where mass profile of the sample of interest is generated and then compared in a large sample population to screen for differences between the samples. When signals that can significantly discriminate between samples are detected, the metabolites are identified and the biological relevance of that compound can be revealed thus reducing the analysis time.

Metabolomics can be used for a large range of applications, including phenotyping of genetically modified plants and substantial equivalence testing, determination of gene function and monitoring responses to biotic and abiotic stress. Metabolomics decreases the gap between genotype and phenotype by providing a more comprehensive view of how cells function, as well as identifying novel or striking changes in specific metabolites. It can provide new hypotheses and new targets for biotechnology.

Metabolic profiling or metabolomicics is referred to as being either targeted or non-targeted:

TARGETED: In the targeted approach, specific metabolites of known identity are profiled. In the case of targeted MS, this involves the addition of multiple stable isotope-labelled standards to the biological sample before the extraction and derivatization steps to control for differences in analyte loss during sample processing and to compensate for ionization-suppression effects. Targeted methods provide an excellent survey of metabolic fuel selection and a profile of energy-yielding metabolic pathways, including elements of mitochondrial metabolism.

NON-TARGETED: In non-targeted profiling it involves the use of NMR or MS for simultaneous measurement of as many metabolites as possible in a biological specimen. These approaches are generally used to compare two biological or clinical states and to report on differences between the two states based on peak areas of raw spectral data.


How has metabolomics contributed to the understanding of disease mechanisms and how has it contributed to the improvement or creating strategies for treatment of these diseases?

  • Human diabetes and insulin resistance

Targeted mass spectrometry (MS) based metabolic profiling has been increasingly applied to studies of human conditions. The profiling of an obese person was done in a research conducted that revealed that branch chain fatty acid catabolism correlates with insulin resistance. The results from this research showed that metabolomics can provide a more detailed picture of metabolic status of normal and pre-diabetic subjects, which can be used for further development and could contribute to more exact sub-classification of different forms of diabetes, leading to the development of more effective drugs.

  • Human cardiovascular disease (CVD)

In a study conducted using MS-based metabolic profiling the application of metabolomics was done to determine metabolic lesions in heart failure and myocardial infarction (MI). The growing number of metabolomics studies in the area of heart failure may ultimately facilitate optimal design of perioperative treatment regimens based on the particular form of cardiovascular disease and the metabolic status of the heart. Comprehensive metabolic profiling, or “metabolomics” is increasingly being applied to CVD, leading to recent discoveries with both form and function implications.

Metabolomics profiling of coronary artery disease (CAD): Targeted MS/MS-based methods were used to profile 45 plasma acylcarnitines and 15 amino acids in a larger study of CAD. With the use of principal components analysis for data reduction, two principal components analysis–derived metabolite factors were found to be associated with CAD: one is composed of branched-chain amino acids (BCAAs) and their associated metabolites and one is composed of urea cycle metabolites, including arginine and citrulline. These metabolite clusters discriminated individuals with CAD from those without CAD in both discovery and validation data sets.

Myocardial infraction:  identified certain proteins such as troponin I, troponin T, C-reactive protein, and B-type natriuretic peptide as diagnostic markers for CVD events and heart failure.

Another recent study has identified a fascinating link between the diet, gut microflora, host metabolism, and metabolomic biomarkers of risk for incident CVD events. The study used a non-targeted LC-MS–based metabolomics approach to profile stable patients who subsequently experienced MI, stroke, or death over the ensuing 3-year period compared with age- and sex-matched control subjects who did not experience events.

Integration of Genetics and Metabolomics for the Identification of Novel Disease Pathways:

 A potential avenue for translating metabolomics-derived biomarkers to disease mechanisms is the integration of metabolomics with other “omics” methods. Human genome-wide association studies have mapped loci associated with polygenic disorders like CVD and diabetes mellitus, but they account for only a small fraction of these diseases and have made limited contributions to knowledge-based therapeutic interventions. This results in part because both conditions are actually a family of diseases in which genetic variability, environmental factors and resultant perturbations in metabolic control within multiple tissues and organs combine to disrupt homeostasis and tissue functions.

Additional Readings:


Nucleic Acids

What is Central Dogma?

central_dogmacentral dogma

The central dogma is the main thesis of molecular inheritance. It states that DNA makes RNA which makes protein. DNA is copied to DNA by the process of DNA replication. The DNA information can be copied into mRNA by the process of transcription. The proteins can now be synthesized using the information in mRNA as a template in the process of translation.

What is a nucleic acid?

It is a naturally occurring chemical compound that is capable of being broken down to yield phosphoric acid, sugars, and a mixture of organic bases (purines and pyrimidines). Nucleic acids are the main information carrying molecules of the cell. It does so by directing the process of protein synthesis and by determining the inherited characteristics of every living thing. The two main classes of nucleic acids are deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). DNA is the master blueprint for life and constitutes the genetic material in all free-living organisms and most viruses. RNA is the genetic material of certain viruses but it is also found in all living cells where it plays an important role in certain processes such as the making of proteins.

What is the difference between a nuclotide and a nucleoside?



Рũßłїѕђёď åŗțїćłё #3

Brouwer, Ingeborg. 2010. ‘Effect of Animal and Industrial Trans Fatty Acids on HDL and LDL Cholesterol Levels in Humans – A Quantitative Review.’ Accessed April 05, 2013.

Effects of Animal and Industrial Trans Fatty Acids on HDL and LDL Cholesterol Levels in Humans- A Quantitative Review:

          Trans fatty acids can be obtained from industrial hydrogenation of vegetable oil and fish oils (artificial trans fatty acids) or from the biohydrogenation from ruminant animals such as cows and sheep (natural trans fatty acids). The consumption of these hydrogenated products results in the increase or decrease of HDL and LDL lipoproteins in the body which places a risk on a person’s heart.

            In this report 39 studies were conducted using persons with controlled diets. Twenty-nine used industrial trans fatty acids, six used ruminant trans fatty acids while seventeen used conjugated trans linoleic acid (CLA). Linear regression analysis was uses to determine if these individuals were affected. The slope of the line for LDL to HDL ration was steeper for trans industrial fatty acids than for ruminant fatty acids or CLA. Statistical analysis was used to compare trans fatty acids with saturated fatty acids.

            The results indicated that there was significant weight loss and gain for some individuals with an increased risk of heart and liver disease. There is a quantitative comparison of the effect of ruminant trans fatty acids and CLA with industrial trans fatty acids on blood lipoproteins in humans. The analysis shows that all three classes of trans fatty acids raise the ratio of LDL to HDL. The effect of ruminant trans fatty acids and CLA on the LDL to HDL ratio was less than that of industrial trans fatty acids. The trans fatty acid with double bonds raised the LDL and lowered the HDL levels of cholesterol.         

            Thus, it was concluded that the removal of all the ruminants trans fatty acids (meat and milk) would lower the total trans fatty acid intake. Further studies need to be conducted to determine if the effects are due to chance. It some countries such as Denmark trans fatty acids are banned from the food industry.  

            This article helped me to better understand trans fatty acids to a larger extent. My knowledge of why trans fatty acids has such a negative impact on our bodies was broadened. Although this substance tantalized our taste buds and increases the shelf life of certain products it is a major component of cholesterol molecules. This as it is known leads to atherosclerosis which leads to heart attacks and strokes.

             I hope that after reading this  blog post you try to change your lifestyle to a more healthier way of living. Remember to exercise regularly, drink 6-8 glasses of water, include a large amount of fresh fruits and vegetables, and keep in mind that what you put into your body will affect you sooner or later.



Understanding Cholesterol…

What is Cholesterol?
Do you know what it is?
Have you ever stopped and wonder how many foods you eat each day that contains this molecule?

Well I am using this video to help you my viewers to understand more about this molecule.
Cholesterol is a waxy, fat like substance found in the blood stream and cells of the body. This naturally occurring substance plays a critical role in the formation of cell membranes and the manufacture of hormones. only a small amount of functions is needed to carry out these functions so the presence of additional cholesterol poses a risk to the body.

How cholesterol works?
This molecule does not dissolve in the blood stream but is transferred in and out of the cell by carriers called low density lipoproteins (LDL) and high density lipoproteins (HDL). When cholesterol increases more lipoproteins is needed to be produced to transfer it across the cell. LDLs are bad because too much of it result in plaque build up in the artery wall which leads to a condition know as atherosclerosis. The arteries are hardened and clogged which can lead to a heart attack or stroke. On the other hand HDL is a good carrier since it aids in the removal of cholesterol from the arteries into the liver and out of the body.

How is cholesterol determined in the body?
A blood test can be done. The levels of cholesterol varies in a person’s age, weight and sex.
An LDL level above 160 is high while an HDL level below 40 is too low. This places someone at risk for plaque build up. 75% of the cholesterol is made in the body while the other 25% is obtained from our diet. It is found in foods such as meat, eggs and liver. Eating less saturated fats from animals is a first step in lowering cholesterol levels and living a healthy life style.

This video was beneficial in helping me understand more about the effects cholesterol. A possible way to better this video is maybe the additional of more pictures to show the effects this molecule has on our body. I enjoyed it sice it was short and to the point.


Рũßłїѕђёď åŗțїćłё #2

 Mattar et al. 2012. ‘Lactose intolerance: diagnosis, genetic and clinical factors.’ Accessed March 28, 2013.

Lactose intolerance: diagnosis, genetic and clinical factors:

Lactose is a carbohydrate found in milk. This disaccharide is made up of glucose and galactose subunits. Seventy five percent of the world’s population loses their ability to breakdown the disaccharide into monosaccharide units that are easily digested. Lactase is the enzyme that breaks down lactose products. In infants breakdown is at its max from birth till 2 years. An aging person can fall into a group of lactase non-persistence (hypolactasia) or lactase-persistence activities. Reduction in lactose renders persons lactose intolerant that develop symptoms in identifying the presence of this diagnosis. 

            Individuals with hypolactasia and lactase persistence have identical coding sequences which were confirmed in a study where DNA was collected from subjects in various parts of the world. The LCT-13910CT and LCT-13910TT genotypes were associated with the lactase-persistence phenotype. This indicates that it dominates the person where they is a lactose digester. If the genotype was LCT- 13910CC and LCT-13910T is absent the person suffers from lactose mal digestion.

            The first method for detection of lactose mal digestion was direct biochemical assay of lactase activity from a jejunal sample. This was performed using a glucose oxidase reagent which detects glucose molecules present in the lactose. This method has been replaced by endoscopic duodenal biopsy. The lactose breath test is also a method for determining the presence of lactose in the body. It is based on fermentation of undigested lactose by intestinal flora producing hydrogen, carbon dioxide and methane which is absorbed and eliminated via the lungs. The result of these gases is bloating, abdominal pain, and diarrhea. Undigested lactose acidifies the colon and increases diarrhea while some may experience constipation.

            A false-negative result can occur if antibiotics have been recently consumed within one month of testing or if the pH is too acidic to inhibit bacterial activity or if there has been bacterial growth. The genetic test provides a more direct result where hypolactasia or lactase persistence genotype is found. This was formed due to the discovery of lactase-persistence alleles. This method was deemed better than the breath test since there is no cut off level or dependence on the amount of lactose or influenced by the duration of the test and age of the individual.

            Individuals suffering with this problem need to maintain their intake of calcium due to their restricted milk diet. A deficient in calcium result in bone diseases. A key to management of lactose intolerance is a recommended of no more than 20g of lactose without significant symptoms. A person’s diet changes where they would have to consume it with other foods and prevent lactose tablets. Supplements of calcium and vitamin D are produced which may be expensive to the consumer.  Yoghurt containing live cultures providing endogenous beta galactosidase is an alternative source of calories and calcium and is well tolerated by many lactose-intolerant patients. Lactose hydrolyzed milk is another safe source for patients.

            This article cleared up the effects of an individual suffering from the absence of the enzyme called lactase which breaks down lactose found in dairy products such as milk. It enhances the symptoms of a patient suffering from lactose intolerance and deals with the different mechanisms of detecting lactose in the body and suggests which method is better due to the information gathered.

            Hope you learned something as well…

Did you know???


Hey guys so I am wrapping up on my Biochemistry blog posts. Today I learnt that cholesterol can actually be beneficial to us humans although we think of it as having this negative impact on our lives. It is a compound of the sterol type, C27H45OH, found in most body tissues and important in metabolism.  It’s structural components is composed of:

  • 4 fused ring collectively referred to as the steroid nucleus
  • An OH group on C3 (hydrophilic polar head)
  • An alkyl side chain located on C17
  • 2 methyl groups on C10 and C13
  • A double bond present between C5 anC6
  • Ring D as seen on the picture above is the only 5 membered ring while the other 3 rings are composed of 6 members

The human body contains about 100 g of cholesterol. Most of this is incorporated in the membranes from which cells are constructed and is an indispensable component of them. The insulating layers of myelin wound around neurons are especially rich in cholesterol.

Other uses of cholesterol include the synthesis of the steroid hormones: progesterone, estrogens, androgens (e.g., testosterone), glucocorticoids (e.g., cortisol) and mineralocorticoids (e.g., aldosterone).

Cholesterol is also the precursor from which the body synthesizes vitamin D.

One of the major uses of cholesterol is the synthesis of bile acids. These are synthesized in the liver from cholesterol and are secreted in the bile. They are essential for the absorption of fat from the contents of the intestine. The liver synthesizes some 1500–2000 mg of new cholesterol each day. It synthesizes cholesterol from the products of fat metabolism.

Cholesterol is seen to be beneficial to the human body. It also has negative impacts on the human body. The health effects of cholesterol problems are due to a condition called atherosclerosis, which is narrowing and hardening of arteries. When  levels of cholesterol are too high, LDLs (low density lipoproteins) will leave extra cholesterol in the blood. If the HDLs (high density lipoproteins) cannot pick up all of this cholesterol it will begin to build up on your artery walls along with other fats and debris. This buildup of cholesterol is called plaque. Over time, plaque can cause narrowing of the arteries or atherosclerosis. Health effects of this process include:

  • High blood pressure – increases over 140/190
  • Heart attack- occurs when the supplyof blood and oxygen to part of the heart is blocked
  • Stroke – it is a sudden occurrence where a blood vessel in the brain gets blocked or ruptures
  • Agina – occurs when the heart is not getting enough oxygen-rich blood for a short time

I hoped you have learnt something that is beneficial in understanding this lipid molecule. Until next time…


Chemiosmotic Hypothesis:


The chemiosmotic hypothesis was developed in 1961 by Peter D. Mitchell which suggests that most ATP synthesis in respiring cells comes from the electrochemical gradient across the inner membranes of mitochondria by using the energy of NADH and FADH2 formed from the breaking down of energy-rich molecules, such as glucose.

The diagram below shows the structure of a mitochondria:

Molecules such as glucose are metabolized through glycolysis to produce acetyl CoA as an energy-rich intermediate. The oxidation of acetyl CoA in the mitochondrial matrix is coupled to the reduction of a carrier molecule such as NAD and FAD. The carriers pass electrons to the electron transport chain (ETC) in the inner mitochondrial membrane, which in turn pass them to other proteins in the ETC. The energy available in the electrons is used to pump protons from the matrix across the inner mitochondrial membrane, storing energy in the form of a trans-membrane electrochemical gradient. The protons move back across the inner membrane through the enzyme ATP synthase. The flow of protons back into the matrix of the mitochondrion via ATP synthase provides enough energy for ADP to combine with inorganic phosphate to form ATP. The electrons and protons at the last pump in the ETC are taken up by oxygen to form water.

Diagram showing the structure of mitochodrial ATP synthase (F1F0 ATPase)

atpase                          pi
In all cells, chemiosmosis involves the PROTON-MOTIVE FORCE (PMF) in some step. This can be described as the storing of energy as a combination of a proton and voltage gradient across a membrane. The chemical potential energy refers to the difference in concentration of the protons and the electrical potential energy as a consequence of the charge separation (when the protons move without a counter-ion).
In most cases the proton motive force is generated by an ETC which acts as both an electron and proton pump, pumping electrons in opposite directions, creating a separation of charge. In the mitochondria, free energy released from the electron transport chain is used to move protons from the mitochondrial matrix to the inter-membrane space of the mitochondria. Moving the protons to the outer parts of the mitochondria creates a higher concentration of positively charged particles, resulting in a slightly positive and slightly negative side. This charge difference results in an electro-chemical gradient. This gradient is composed of both the pH gradient and the electrical gradient. The pH gradient is a result of the H+ ion concentration difference. Together the electro-chemical gradient of protons is both a concentration and charge difference and is often called the proton motive force. The PMF needs to be about 50 kJ/mol for the ATP synthase to be able to make ATP.
CHEMIOSMOTIC PHOSPHORYLATION is the third pathway that produces ATP from inorganic phosphate and an ADP molecule. This process is part of OXIDATIVE PHOSPHORYLATION. The complete breakdown of glucose in the presence of oxygen is called cellular respiration. The last steps of this process occur in mitochondria. The reduced molecules NADH and FADH2 are generated by the Krebs cycle and glycolysis. These molecules pass electrons to an electron transport chain, which uses the energy released to create a proton gradient across the inner mitochondrial membrane. ATP synthase then uses the energy stored in this gradient to make ATP. This process is called oxidative phosphorylation because oxygen is the final electron acceptor and the energy released by reducing oxygen to water is used to phosphorylate ADP and generate ATP.

g v b


Unsaturated Fats:

characteristics include:

  • at least one carbon-carbon double bond

  • liquid at room temperature

  • kinks created which prevents packing

  • contain cis double bonds

  • monounsturated chain contains only one double bond eg. oleic acid

  • polyunsaturated fats contain more than one double bond eg. linoleic acid

  • sources are plants and fish fats



Saturated Fats:

characteristics include:

  • no carbon-carbon double bond

  • solid at room temperature

  • no kinks created so there is tightly packing

  • long, straight chains

  • eg. stearic acid and palmitic acid

  • sources are from animal fats



Trans Fats:

It seemed like such a good thing once since it enhances the flavor, texture and shelf life of many processed foods. However, it comes with a health risk. Trans fatty foods tantalize a persons taste buds then travel through your digestive system to your arteries where they turn to sludge.

Small amounts of trans fats occur naturally in beef, lamb and full-fat dairy products. Most come from processing liquid vegetable oil to become a solid fat through a process called hydrogenation.